PROJECT SUMMARY/ABSTRACT The process through which mRNAs are selected for translation into proteins, or translation initiation, plays a major role in gene expression, and regulation of this process is critical in diseases such as cancer, diabetes, and neurological diseases. Motivated by this, the long-term goal of our studies is to understand and modulate non-canonical initiation mechanisms that play a significant role in the biology of these diseases. For most cellular mRNAs, initiation involves recognition of the N7-methylguanosine-triphosphate `cap' at the 5' end of the mRNA by eIF4E, the cap-binding protein, an event that results in the recruitment of additional eIFs, including eIF4G, and, ultimately, a ribosomal pre-initiation complex (PIC) that can initiate translation of the mRNA. Remarkably, under cellular stress conditions, such as tumor hypoxia, viral infection, nutrient limitation, etc., where the ability of eIF4E to recruit eIF4G and, consequently, cap-dependent initiation are compromised, a subset of mRNAs encoding genes that aid survival under such stress conditions can still be translated efficiently. These mRNAs somehow switch from the canonical, cap-dependent initiation pathway to a non-canonical, cap-independent initiation pathway. The mechanisms that regulate switching between cap- dependent and cap-independent initiation of these mRNAs remain unknown, impeding our understanding and ability to modulate this aspect of human health and disease. Previous studies of a candidate set of mRNAs, HIF-1?, FGF-9, VEGF-A and p53, that can switch from cap-dependent to cap-independent initiation as a result of eIF4E inhibition, have shown that switching correlates with increased levels of eIF4G or its homolog, DAP5 (eIF4G/DAP5). Interestingly, these mRNAs have also been shown to contain highly structured 5' untranslated regions that act as translational enhancers (TEs) that might play a role in regulating the switch between cap-dependent and cap-independent initiation. Based on our strong preliminary data, we hypothesize that the TEs in TE-containing mRNAs (TE-mRNAs) regulate this switch by directly recruiting eIF4G/DAP5, thereby enabling TE-mRNAs to recruit PICs even when eIF4E is inhibited. Guided by our preliminary data and using ensemble and single-molecule fluorescence methods in combination with complementary cellular and molecular biology approaches, we propose to: (1) characterize the strength and selectivity of eIF4G/DAP5 binding to TE-mRNAs, (2) determine the structural basis for TE recognition by eIF4G/DAP5, and (3) elucidate the causal relationship between the binding of eIF4G/DAP5 to the TEs of TE-mRNAs and the efficiency of PIC recruitment and translation of these TE-mRNAs. Collectively, the studies proposed here will broadly impact the field by providing a model through which an essential subset of mRNAs, TE-mRNAs, switch between a canonical and a non-canonical mechanism of initiation. The results of the proposed studies may provide insights into the mechanism of cap- dependent initiation as well as the mechanisms of other cap-independent initiation pathways.